Immunoassay for the quantitation of human C4 gene products

Abstract

Utilizing mouse monoclonal antibodies which recognize Rodgers 1 and Chido 1 epitopes carried on the C4A and C4B molecules, and heat aggregated IgG to activate C1, an immunoassay was developed for the quantitation of complement components, including total C4, C4A and C4B. Interassay variation was 12.4%, 11.5% and 10.8%, respectively. The immunoassay was compared to the quantitation of total C4 by radial immunodiffusion by testing 103 random white controls and gave a Pearson's product moment correlation coefficient of 0.81. Three genetic total C4 deficient individuals were nonreactive in all three assays. This activated assay is specific, reproducible and superior to existing methods for the quantitation of C4A and C4B and detection of the heterozygous C4 null states.

What is claimed is: 
     
       1. An assay method for C4 comprising the steps of: a) obtaining a preparation of aggregated immunoglobulin molecules;   b) affixing said immunoglobulin to a solid matrix;   c) exposing said immunoglobulin to a sample to be assayed for C4 under such conditions that said C4 will bind to said immunoglobulin; and   d) exposing said bound C4 to an antibody that specifically binds to C4 under conditions such that binding between C4 and said antibody occurs; and   e) detecting the antibody bound.   
     
     
       2. An assay method for C4 comprising the steps of: a) obtaining a preparation of aggregated immunoglobulin molecules;   b) affixing said immunoglobulin to a solid matrix;   c) exposing said immunoglobulin to a sample to be assayed for C4 in the presence of C1 under such conditions that said matrix-bound immunoglobulin will cause capture of C4; and `d) exposing said captured C4 to an antibody that specifically binds to C4 under conditions such that binding between C4 and said antibody occurs; and   e) detecting the antibody bound.   
     
     
       3. The method of claim 1 or claim 2 wherein said immunoglobulin comprises IgG. 
     
     
       4. The method of claim 1 or claim 2 wherein said immunoglobulin comprises IgM. 
     
     
       5. The method of claim 1 or claim 2 wherein said antibody is coupled to a labeling molecule and step e) is performed by detecting said label. 
     
     
       6. The method of claim 5 wherein said label is selected from the group consisting of: fluorescent labels and radioactive labels. 
     
     
       7. The method of claim 1 wherein said antibody is coupled to an enzyme capable of cleaving a selected substrate to produce a chromophore and step e) is performed by adding said substrate so that it is cleaved by said enzyme to produce said chromophore and detecting said chromophore. 
     
     
       8. The method of claim 1 wherein said antibody is detected with a secondary reagent capable of specifically binding to said antibody and said secondary reagent is labeled. 
     
     
       9. The method of claim 8 wherein said secondary reagent is an antibody. 
     
     
       10. The method of claim 1 or claim 2 wherein said antibody is a monoclonal antibody specific for a Rodgers antigen. 
     
     
       11. The method of claim 1 or claim 2 wherein said antibody is a monoclonal antibody specific for a chido antigen. 
     
     
       12. An assay method for C4 comprising the steps of: a) obtaining a solid matrix having aggregated immunoglobulin affixed thereto;   b) exposing said immunoglobulin to a sample to be assayed for C4 under such conditions that said C4 will bind to said immunoglobulin; and   c) exposing said bound C4 to an antibody that specifically binds to C4 under conditions such that binding between C4 and said antibody occurs; and   d) detecting the antibody bound.   
     
     
       13. An immunoassay kit adapted for use in a method for detecting C4 in a biological sample by exposing the sample in the presence of C1 to heat-aggregated, ultrasonication-aggregated, or chemically-aggregated immunoglobulin bound to a solid matrix so that C4 becomes affixed to said matrix and is detected by an antibody directed against C4, which kit comprises: a) a first container containing the heat-aggregated, ultrasonication-aggregated, or chemically-aggregated immunoglobulin reagent; and   b) a second container containing the antibody directed against C4; said aggregated immunoglobulin reagent and antibody being present in a quality and quantity which renders said kit suitable for use in said method.     
     
     
       14. The kit of claim 13 wherein said first container comprises the well of a cell culture dish and said immunoglobulin is bound to said well. 
     
     
       15. The kit of claim 13 further comprising a container comprising a selected concentration of C4. 
     
     
       16. The kit of claim 13 further comprising a container comprising C1. 
     
     
       17. The kit of claim 13 wherein said antibody is labeled with an enzyme capable of cleaving a substrate to produce a chromophore; and said kit further comprises a container comprising said substrate. 
     
     
       18. An immunoassay kit adapted for use in a method for detecting C4 in a biological ample by exposing the sample in the presence of C1 to heat-aggregated, ultrasonication-aggregated, or chemically-aggregated immunoglobulin bound to a solid matrix so that C4 is affixed to said matrix and is detected by an antibody directed against C4, which kit comprises: a) a first container containing the heat-aggregated, ultrasonication-aggregated, or chemically-aggregated immunoglobulin reagent; and   b) a second container containing the antibody directed against C4; and   c) a third container containing a positive control reagent containing C1 and C4; aggregated immunoglobulin reagent, antibody, and positive control reagent being present in a quality and quantity which renders said kit suitable for use in said method.